Wiebke Hoth-Hannig

The effect of acidic beverages on the ultrastructure of the acquired pellicle—An in situ study

AIM The aim of the present in situ study was to investigate ultrastructural alterations as well as protective properties of the pellicle layer during consumption of acidic beverages. METHODS Bovine enamel slabs were fixed on buccal and palatal aspects of individual splints and exposed in the oral cavities of three subjects for 120 min. In the following, the subjects drank orange juice, coke light or sprite light. Half of the specimens were removed afterwards, the others were exposed to the oral fluids for another 120 min. Erosive alterations of the bovine enamel slabs were measured by determination of the Knoop-micro-hardness. In addition, the ultrastructure of the pellicle was evaluated by transmission electron microscopy (TEM). RESULTS Determination of Knoop-micro-hardness yielded only little reduction of the relative Knoop-hardness in situ during consumption of sprite light (-0.053+/-0.019) and coke light (-0.075+/-0.04). With orange juice nearly no change of the hardness was recorded. TEM-pictures showed that the globular outer layers of the pellicle were removed to a different extent according to the localisation of the specimens in the oral cavity, whereas the basal pellicle was not affected by the acidic beverages. On the specimens carried for another 120 min after the erosive attack, lacunae filled with organic structures were observed underneath the basal side of the pellicle. CONCLUSION During fast consumption of acidic beverages in situ, the erosive effects on pellicle coated bovine enamel are moderate and juices seem to be less harmful as compared with low pH soft drinks. Pellicle proteins in eroded lacunae may impact the remineralization process.

Initial bioadhesion on dental materials as a function of contact time, pH, surface wettability, and isoelectric point.

The adsorption of bovine serum albumin (BSA) on surfaces of dental enamel and of dental materials was investigated by scanning force spectroscopy. This method provides adhesion forces which can be measured as a function of contact time between protein and surface, pH, wettability, and isoelectric point of the surface. Whereas the chosen ceramic and composite materials resemble very well the adhesion on natural enamel, a much stronger adhesion was found for the more hydrophobic surfaces, that is, gold, titanium, poly(methyl methacrylate) (PMMA), and poly(tetrafluoroethylene) (PTFE). On hydrophilic surfaces, adhesion is mainly influenced by the electrostatic forces between protein and surface. However, the conformational change of BSA at pH values above pH 8 has to be taken into account. On the very hydrophobic PTFE surface, the special interface structure between PTFE and water plays an important role which governs BSA adhesion.

Transmission electron microscopy comparison of methods for collecting in situ formed enamel pellicle

The in vivo formed salivary pellicle is composed of an outer globular and a densely structured basal layer. This study developed a method for selective recovering of these pellicle layers from the enamel surface. Two-hour in situ pellicles were formed by intraoral exposure of enamel specimens in two adults. Pellicle-covered enamel specimens were treated either mechanically (scraping with scaler, curette or razor blade, or rubbing with a sponge) or chemically (phosphate buffer, NaCl, NaOCl, CaCl2, NaSCN, urea, tetrahydrofurane, guanidine, SDS, HCl, or EDTA with or without additional ultrasonication). Specimens were processed for transmission electron microscopic analysis to detect pellicle residues remaining on the enamel surface after the different treatments. Most of the chemical treatments caused partial, incomplete removal of the globular layer. Complete removal of the globular layer without disruption of the basal layer was obtained by sponge rubbing or by CaCl2 combined with ultrasonication, whereas scraping caused partial disruption of the basal layer. Removal of the basal layer was observed after treatment with HCl, EDTA, or NaOCl combined with ultrasonication. Electrophoretical analysis of recovered pellicle fractions indicate that combination of sponge-rubbing followed by EDTA treatment can be recommended for stepwise removal of the globular and basal pellicle layers.

Electron microscopic detection and activity of glucosyltransferase B, C, and D in the in situ formed pellicle.

OBJECTIVE Glucosyltransferases (GTFs) represent a virulence factor of mutans streptococci. The aim of the present in situ study was to investigate the distribution of different GTF-isoforms in the pellicle. DESIGN Bovine enamel slabs were fixed on buccal and palatal sites of individual splints worn by five subjects for 30 and 120 min to allow pellicle formation. Pellicle specimens were processed for transmission electron microscopy (TEM) and field emission in-lens scanning electron microscopy (FEI-SEM). Gold-immunolabelling was used for detection of GTF-isoforms B, C and D. Furthermore, glucosyltransferase activity of 3-, 30- and 120-min pellicles was tested via determination of fructose release. RESULTS All isoforms of the enzyme were found to be randomly distributed within all layers of the pellicle. In cross-sections (TEM), GTF D was the most abundant isoform. More labelled molecules were detected on buccal sites compared with palatal surfaces, the number of molecules detected increased with time. The amount of GTF B, C and D found on the pellicle surface by FEI-SEM showed no correlation with pellicle formation time or localisation in the oral cavity. Overall, GTF D was detected more frequently on the surface than GTF B and C. All pellicles tested showed GTF-activity. CONCLUSION The study shows for the first time the presence of the GTF-isoforms B, C and D within all layers of the in situ formed pellicle. This emphasises the impact of streptococcal products on the composition of the pellicle and illustrates a mechanism used by bacteria to colonize dental surfaces.

Effect of Safflower Oil on the Protective Properties of the in situ Formed Salivary Pellicle

Aim: The prevalence of dental erosion is still increasing. A possible preventive approach might be rinsing with edible oils to improve the protective properties of the pellicle layer. This was tested in the present in situ study using safflower oil. Methods: Pellicle formation was carried out in situ on bovine enamel slabs fixed buccally to individual upper jaw splints (6 subjects). After 1 min of pellicle formation subjects rinsed with safflower oil for 10 min, subsequently the samples were exposed in the oral cavity for another 19 min. Enamel slabs without oral exposure and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After pellicle formation in situ, slabs were incubated in HCl (pH 2; 2.3; 3) for 120 s, and kinetics of calcium and phosphate release were measured photometrically (arsenazo III, malachite green). Furthermore, the ultrastructure of the pellicles was evaluated by transmission electron microscopy (TEM). Results: Pellicle alone reduced erosive calcium and phosphate release significantly at all pH values. Pellicle modification by safflower oil resulted in an enhanced calcium loss at all pH values and caused an enhanced phosphate loss at pH 2.3. TEM indicated scattered accumulation of lipid micelles and irregular vesicle-like structures attached to the oil-treated pellicle layer. Acid etching affected the ultrastructure of the pellicle irrespective of oil rinsing. Conclusion: The protective properties of the pellicle layer against extensive erosive attacks are limited and mainly determined by pH. The protective effects are modified and reduced by rinses with safflower oil.

Protein adhesion on dental surfaces—a combined surface analytical approach

Protein adsorption is a field of huge interest in a number of application fields. Information on protein adhesion is accessible by a variety of methods. However, the results obtained are significantly influenced by the applied technique. The objective of this work was to understand the role of adhesion forces (obtained by scanning force spectroscopy, SFS) in the process of protein adsorption and desorption. In SFS, the protein is forced to and retracted from the surface, even under unfavorable conditions, in contrast to the natural situation. Furthermore, adhesion forces are correlated with adhesion energies, neglecting the entropic part in the Gibbs enthalpy. In this context, dynamic contact angle (DCA) measurements were performed to identify the potential of this method to complement SFS data. In DCA measurements, the protein diffuses voluntarily to the surface and information on surface coverage and reversibility of adsorption is obtained, including entropic effects (conformational changes and hydrophobic effect). It could be shown that the surface coverage (by DCA) of bovine serum albumin on dental materials correlates well with the adhesion forces (by SFS) if no hydrophobic surface is involved. On those, the entropic hydrophobic effect plays a major role. As a second task, the reversibility of the protein adsorption, i.e., the voluntary desorption as studied by DCA, was compared to the adhesion forces. Here, a correlation between low adhesion forces and good reversibility could be found as long as no covalent bonds were involved. The comparative study of DCA and SFS, thus, leads to a more detailed picture of the complete adsorption/desorption cycle.

Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle

Objectives: In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. Methods: The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4′,6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicles ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). Results: Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. Conclusion: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Influence of Calcium Phosphate and Apatite Containing Products on Enamel Erosion

For the purpose of erosion prevention the present study aimed to compare the efficacy of two biomimetic products and a fluoride solution to optimize the protective properties of the pellicle. After 1 min of in situ pellicle formation on bovine enamel slabs, 8 subjects adopted CPP-ACP (GC Tooth Mousse), a mouthwash with hydroxyapatite microclusters (Biorepair), or a fluoride based mouthwash (elmex Kariesschutz) for 1 min each. Afterwards, samples were exposed in the oral cavity for 28 min. Native enamel slabs and slabs exposed to the oral cavity for 30 min without any rinse served as controls. After oral exposure, slabs were incubated in HCl (pH values 2, 2.3, and 3) for 120 s and kinetics of calcium and phosphate release were measured photometrically; representative samples were evaluated by SEM and TEM. The physiological pellicle reduced demineralization at all pH values; the protective effect was enhanced by fluoride. The biomimetic materials also reduced ion release but their effect was less pronounced. SEM indicated no layer formation after use of the different products. However, TEM confirmed the potential accumulation of mineral components at the pellicle surface. The tested products improve the protective properties of the in situ pellicle but not as effectively as fluorides.

Do phosphoric acid esters affect the immediate enamel bond strengths

Abstract Purpose: To investigate the effects of phosphoric acid esters (PAEs)-containing primers on the micro-tensile bond strengths (MTBS) of etched enamel, the micro-morphologies of the resin–enamel interfaces, and the enamel surfaces. Materials and Methods: Thirty-three bovine incisors were used in this study. After the labial enamel surfaces were highly polished, they were etched, water sprayed, and air-dried. Afterward, the enamel surfaces were treated with or without (control) one of three PAEs-containing primers (40%MDP, Ivoclar Vivadent; Resulcin AquaPrime A + B, DMG; Xeno V, Dentsply), water sprayed, and air-dried. Subsequently, an adhesive Heliobond (Ivoclar Vivadent) was applied and resin composite (Core Build-up, Bisco) was placed incrementally. They were prepared into multiple beams of about 1 × 1 × 8 mm for MTBS tests. The enamel surfaces and resin–enamel interfaces were analyzed by SEM and TEM. The data were analyzed by two-way ANOVA and LSD test. Results: Using PAEs-containing primers could increase the enamel MTBS (p < 0.05). The primer MDP produced higher MTBS than the primer Resulcin AquaPrime A + B and Xeno V. The SEM findings revealed the primed enamel surfaces were covered with a thin or glue-like layer of monomer-calcium salts of PAEs and the residual enamel crystallites, and various micro-porosities were detected within the hybrid layers. The TEM findings revealed the hydroxyapatite crystallites were tightly covered by the adhesive, the adhesive mixed with resin composite, or sparse irregular enamel crystallites. Conclusion: The application of PAEs-containing primers on the etched enamel substrate could significantly increase the immediate enamel bond strengths. However, this effect was dependent on the individual PAEs-containing primer used.